Metal ions are very useful as reporter substances. They can be detected in very low concentrations by emission of radioactivity, fluorescence, electron spin resonance and NMR relaxation. When attached to a carrier molecule, such as an antibody or an antigen, they can report its concentration or location. The carrier molecule may have an avidity for disease associated molecules, e.g., tumor-associated antigens. Thus, carrier molecules conjugated to metal ions can be used as in vivo and in vitro diagnostic tools to detect the presence and/or location of disease in the body. Certain radiation-emitting metal ions may also be attached to carrier molecules, such as antibodies to tumor cell surface antigens, for the purpose of carrying the metal ions to a tumor site in order to irradiate the tumor.
Problems may be encountered when attempting to label certain carrier molecules with some metal ions. Conditions which stabilize the metal ion may not be compatible with stability of the carrier molecule and vice versa. An example of such incompatibility occurs in the labeling of a protein or polypeptide with indium-111 or gallium-67. Metal ions can be joined to a protein or polypeptide by producing a derivative of the protein or polypeptide which contains a moiety having a terminal chelating group capable of forming a chelate with the metal ion. For example, my copending U.S. patent application Ser. No. 650,127 describes a method of joining a radionuclide metal ion to an antibody fragment using a bifunctional coupling agent which reacts with a free sulfhydryl group of the antibody fragment and also contains a chelating group capable of complexing a metal ion. U.K. Patent Application GB No. 2 109 407 also describes the use of chelate-derivatized antibodies to form antibody-metal ion conjugates for use in tumor imaging. When labeling chelate-derivatized proteins with indium-111 or gallium-67 two problems are encountered. These metal ions are used in the form of their chloride salts, which must be kept in aqueous solutions at a pH less than 3.5. If they are not, they will react with water to form insoluble metal hydroxides. This acidic pH may adversely affect the stability or biological activity of the protein or polypeptide.
Technetium presents an additional set of problems for protein labeling. Technetium exists in 7 oxidation states, the most stable one being +7 as in pertechnetate (TcO.sub.4.sup.-). The +5 state is very useful for labeling chelates and chelate coupled proteins. However, this oxidation state is not easily achieved. Being metastable, technetium (+5) is easily reduced to (+4), and trapped as reduced-hydrolyzed TcO.sub.2 in the presence of excess reducing agent. Reduced-hydrolyzed technetium is not useful for chelate or protein labeling, because of its tendency to self-associate or bind non-specifically to surfaces. If stoichiometric amounts of reducing agents are used, reduction is kinetically very inefficient. The +5 technetium so generated has a tendency to reoxidize to pertechnetate as soon as the reducing agent is consumed.
Other problems are encountered in forming chelate complexes because of functional groups on the protein or polypeptide molecules. The amino and carboxyl terminus, the amide bond backbone and the side chain residues of aspartic and glutamic acid, lysine, cysteine, tyrosine and histidine found in proteins and polypeptides all possess chelate ligand character. The amino acid sequence and tertiary structure of the molecule can bring these chelate ligands together to create strong, multidentate metal ion binding sites. Thus, each protein possesses a spectrum of metal binding properties. The weak sites can leach the metal ion in the blood stream to other plasma proteins or small molecular weight constituents. The strong sites interfere with protein metabolism. This invariably leads to radionuclide retention in major protein catabolic sites such as the liver and kidney.
One particularly troublesome problem encountered with technetium labeling of antibodies during in situ reduction of pertechnetate is the concomitant reduction of protein disulfide bonds. The resulting sulfhydryl groups bind technetium in preference to coupled chelates (Paik, C.H. et al., J. Nucl. Med. Biol., 12:3 [1985]). The resulting labeled antibody yields metabolites that do not clear from liver or kidney. This is an ideal property for technetium labeled microaggregated albumin for imaging normal liver, but it is highly undesirable for labeled antibodies used for in vivo diagnostic applications, where clearance of the radionuclide from the liver is desired.
The magnitude of this clearance problem can be appreciated if one considers the metabolic fate of labeled antibodies. A consistent observation in the use of labeled antibodies for tumor detection is that only a small fraction of the injected dose goes to the target solid tumor (Larson, S.M., et al., J. Clin. Invest., 72:2101-2114 (1983); Buraggi, G.L., et al., Cancer Res., 45:3378-3387 (1985); Reviewed in: Halpern, S.E., et al., Diagnostic Imaging, June, 40-47 (1983)). This is because the tumor has a relatively low blood flow and vascular permeability. Thus, the tumor has a very low labeled antibody extraction efficiency in comparison to competing catabolic pathways of labeled antibody uptake. Under optimal conditions, a target tissue will take up from 3-10% of the injected dose. More often, tumor uptake is less than 1%. The remainder, 90% or more, must be processed by the liver and kidneys. Antibodies labeled with radionuclide metal ions by procedures of the prior art do not provide rapid washout of the radiolabeled catabolites from these sites. There are two adverse consequences of this radionuclide retention. The high level of radiation being emitted from the liver or kidneys obscures the low level of radiation coming from the target tumor. More importantly, the liver and kidneys, being among the most radiosensitive organs, accumulate an unacceptable dose of radiation. Dosimetry is linearly dependent on label concentration, but exponentially dependent on time of exposure. Therefore, decreasing the liver and kidney exposure time from no clearance to a fraction of the radionuclide's half-life will yield a dramatic decrease in radiation dose.
It would be highly desirable to devise an antibody labeling method that causes a radionuclide to be retained on the surface of the target tumor cells for at least two half-lives, but yields labeled metabolites which have biological half-lives of less than 60 minutes in normal clearance organs.
One method of labeling a chelate-derivatized protein or polypeptide, such as an antibody, involves the use of a so-called "transfer ligand." A transfer ligand is a compound which forms an intermediate complex with the metal ion. The metal ion complex with the transfer ligand is soluble under conditions in which the protein is stable. The complex between the transfer ligand and the metal ion is such that the metal ion is transferred upon contact with the chelate-derivatized protein which has a stronger chelating affinity for the metal ion than does the transfer ligand. European Patent Application No. 82201602.8 describes a method of labeling proteins with radionuclide metal ions which employs an intermediate complex of the metal ion in the form of a carboxylate, dithiocarboxylate, enolate or mixture thereof.
For various reasons, the transfer ligands employed in the prior art have not been completely satisfactory. Ideally, a transfer ligand possesses a unique profile of properties. For use in conjunction with radionuclides having stable oxidation states, such as indium-111 and gallium-67, these properties are as follows:
1. It prevents the radionuclide metal ion from forming metal hydroxides in physiologic buffers at neutral pH;
2. It prevents the radionuclide metal ion from binding to moderately strong endogenous chelating ligands of native or chelate-derivatized proteins;
3. It transfers the radionuclide metal ion rapidly and quantitatively to strong chelating groups that have been coupled to the protein;
4. It is water soluble, non-toxic and non-mutagenic; and
5. It is readily available and, preferably, inexpensive.
For use in conjunction with oxidation-sensitive radionuclides, such as technetium, the transfer ligand should also possess the following properties:
6. It quantitatively traps the radionuclide metal ion in the proper oxidation state during the reduction process for subsequent chelation; and
7. It prevents the reoxidation of the radionuclide metal ion when the reducing agent is removed.
To meet these criteria, a transfer ligand must possess two physicochemical properties. Its complex with the radionuclide ion must be kinetically labile, permitting rapid exchange when it is presented with a more thermodynamically stable multidentate ligand. Additionally, the transfer ligand must have sufficient thermodynamic stability to prevent exchange with hydroxide ions or endogenous chelating sites on protein molecules in aqueous buffers at physiologic pH. While the former property is generally characteristic of metal ion indicators used in ethylenediaminetetraacetic acid complexometric titrations, very few of the metal ion indicators or the weak chelating agents (such as those described in European Patent Application No. 82201602.8) meets all seven of the criteria listed above for an ideal transfer ligand.